- Email: [email protected]
Short leukocyte telomere length predicts poor prognosis and indicates altered immune functions in colorectal cancer patients
original articles
Annals of Oncology 21. Verwaal VJ, van Ruth S, Witkamp A et al. Long-term survival of peritoneal carcinomatosis of colorectal origin. Ann Surg Oncol 2005; 12: 65–71. 22. Younan R, Kusamura S, Baratti D et al. Morbidity, toxicity, and mortality classification systems in the local regional treatment of peritoneal surface malignancy. J Surg Oncol 2008; 98: 253–257. 23. Cao C, Yan TD, Black D et al. A systematic review and meta-analysis of cytoreductive surgery with perioperative intraperitoneal chemotherapy for peritoneal carcinomatosis of colorectal origin. Ann Surg Oncol 2009; 16: 2152–2165. 24. Oosterling SJ, van der Bij GJ, van EM et al. Surgical trauma and peritoneal recurrence of colorectal carcinoma. Eur J Surg Oncol 2005; 31: 29–37. 25. Raa ST, Oosterling SJ, van der Kaaij NP et al. Surgery promotes implantation of disseminated tumor cells, but does not increase growth of tumor cell clusters. J Surg Oncol 2005; 92: 124–129.
26. van den Tol MP, ten RS, van Grevenstein WM et al. The post-surgical inflammatory response provokes enhanced tumour recurrence: a crucial role for neutrophils. Dig Surg 2007; 24: 388–394. 27. Gur I, Diggs BS, Wagner JA et al. Safety and outcomes following resection of colorectal liver metastases in the era of current perioperative chemotherapy. J Gastrointest Surg 2013; 17: 2133–2142. 28. Nordlinger B, Sorbye H, Glimelius B et al. Perioperative FOLFOX4 chemotherapy and surgery versus surgery alone for resectable liver metastases from colorectal cancer (EORTC 40983): long-term results of a randomised, controlled, phase 3 trial. Lancet Oncol 2013; 14: 1208–1215. 29. Passot G, Vaudoyer D, Cotte E et al. Progression following neoadjuvant systemic chemotherapy may not be a contraindication to a curative approach for colorectal carcinomatosis. Ann Surg 2012; 256: 125–129.
Annals of Oncology 25: 869–876, 2014 doi:10.1093/annonc/mdu016 Published online 10 March 2014
Y. Chen1, †, F. Qu1, †, X. He2, G. Bao2, X. Liu3, S. Wan4 & J. Xing1* 1 State Key Laboratory of Cancer Biology and Experimental Teaching Center of Basic Medicine, Fourth Military Medical University, Xi’an; 2Department of General Surgery, Tangdu Hospital, Fourth Military Medical University, Xi’an; 3Deparment of Gastroenterology, Xijing Hospital of Digestive Disease, Fourth Military Medical University, Xi’an; 4 Pharmaceutical College, Henan University, Kaifeng, People’s Republic of China
Received 15 October 2013; revised 19 December 2013; accepted 30 December 2013
Background: Numerous studies indicate that the leukocyte telomere length is associated with the risk of cancers, including colorectal cancer (CRC). However, the prognostic value of leukocyte telomere length in CRC patients has not been investigated. Patients and methods: Relative telomere length (RTL) of peripheral blood leukocytes (PBLs) from 571 CRC patients receiving surgical resection was measured using a polymerase chain reaction-based method. The Cox proportional hazards ratio model and the Kaplan–Meier curve were used to estimate the association between RTL and the clinical outcome of CRC patients in the training set (90 patients) and the testing set (86 patients). Finally, an independent cohort of 395 patients was used as an external validation set. The immunophenotype of PBLs and the plasma concentration of several immune-related cytokines were determined by flow cytometry and enzyme-linked immunosorbent assay, respectively. Results: Patients with shorter RTL had significantly poorer overall survival and relapse-free survival than those with longer RTL in the training, testing and validation sets. Furthermore, leukocyte RTL and Tumor-Node-Metastasis (TNM) stage exhibited a significant joint effect in the prognosis prediction of combined CRC patients, indicating that patients with both short RTL and advanced stages had the worst prognosis, when compared with other subgroups. In addition, patients with short RTL showed the higher percentage of CD4+ T cell and the lower percentage of B cell in peripheral blood mononuclear cells, as well as the lower concentration of plasma transforming growth factor-β1, suggesting a possibility that the immune functions changed with RTL alteration. Conclusions: Our study for the first time demonstrates that leukocyte RTL is an independent prognostic marker complementing TNM stage and associated with the immune functions in CRC patients.
*Correspondence to: Dr Jinliang Xing, State Key Laboratory of Cancer Biology and Experimental Teaching Center of Basic Medicine, Fourth Military Medical University; 169 Changle West Road, Xi’an 710032, People’s Republic of China. Tel: +86-29-84774764; Fax: +86-29-84774764; E-mail: [email protected] †
YC and FQ contributed equally to this work.
© The Author 2014. Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved. For permissions, please email: [email protected].
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Short leukocyte telomere length predicts poor prognosis and indicates altered immune functions in colorectal cancer patients
original articles
Annals of Oncology
Key words: colorectal cancer, cytokine, immunophenotype, leukocyte relative telomere length, overall survival, relapse-free survival
introduction
patients and methods patients and samples A total of 571 eligible patients with primary colorectal adenocarcinoma were enrolled from two independent sites, Xijing Hospital and Tangdu Hospital, Xi’an, China. All patients received surgical resection and had no preoperative anticancer treatment. Patient subgrouping was described in Supplementary Methods, available at Annals of Oncology online. Twenty-four additional CRC patients were enrolled from Xijing Hospital for immunoassays. Before surgery, 5 ml of venous blood sample was collected from each patient. The
| Chen et al.
measurement of RTL by real-time polymerase chain reaction Leukocyte genomic DNA was extracted from blood samples using the RelaxGene Blood DNA System (TIANGEN, Beijing, China) according to the manufacturer’s instructions. RTL was measured using a real-time polymerase chain reaction-based method detailedly described in Supplementary Methods, available at Annals of Oncology online.
determination of lymphocyte immunophenotype by flow cytometery Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation over Ficoll-Hypaque (Amersham Pharmacia Biotech, NJ, USA) from venous blood of additional 24 CRC patients. The cells were then fixed and stained with isotype control immunoglobulins or fluorescenceconjugated antibodies against the following immune markers: CD3, CD4, CD8, CD25, FOXP3, CD19 and CD56 (eBioscience, San Diego, CA, USA). Finally, the subtype analysis of PBMCs was carried out on a FACScan flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA).
cytokine detection by enzyme-linked immunosorbent assay Blood plasma from the same 24 CRC patients was isolated by centrifugation at 2800 ×g under 4°C. The concentrations of interleukin (IL)-2, IL-4, transforming growth factor (TGF)-β1, tumor necrosis factor (TNF)-α and interferon (IFN)-γ were examined by enzyme-linked immunosorbent assay (ELISA) using commercial kits (eBioscience) according to the manufacturer’s instructions.
statistical analysis The difference of variables among subgroups was compared by the corresponding statistical methods. More details were described in Supplementary Methods, available at Annals of Oncology online. Receiver operating characteristic (ROC) curve was used to calculate the optimal cutoff point of RTL for prognostic prediction in the training set as described previously [11]. The multivariate Cox proportional hazards regression model and the Kaplan–Meier survival curve were used for prognosis evaluation. All statistical analyses were performed using the SPSS Statistics 19.0 software (IBM), and P < 0.05 was considered statistically significant.
results characteristics and RTL distribution of CRC patients The clinical characteristics of the study populations were summarized in Supplementary Table S1, available at Annals of Oncology online. Overall, there were 361 patients who received standard fluorouracil (FU)-based adjuvant chemotherapy (98 with CapeOX regimen, 252 with FOLFOX regimen and 11 with capecitabine single-treatment). One hundred and fifty patients died of CRC and 186 developed relapse during the follow-up period (median, 28 months; range, 6–60 months).
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Colorectal cancer (CRC) is one of the most common malignancies worldwide, with over 600 000 deaths each year [1]. In recent years, increased incidence and death rate of CRC is observed in several regions where this disease has previously been reported to be at low risk. Tumor-Node-Metastasis (TNM) staging system is widely used to assist the decision-making of treatment and prognosis predicting in CRC patients [2]. However, due to the molecular and genetic heterogeneity, TNM stage alone is not sufficient to accurately predict the clinical outcomes of CRC patients. Therefore, it is urgent to explore new biomarkers to complement TNM staging system for better prediction of the CRC prognosis. Telomere is a specified structure consisting of tandem repeats of TTAGGG which caps the end of linear chromosomes in eukaryote cells [3]. Telomere plays a critical role in prevention of genomic instability and DNA shortening-induced damage responses. Under physiological conditions, most somatic cells suffer from gradually shortening of telomere by 15–50 bp each year due to the lack of telomerase activity [4]. Abnormal alteration of telomere length causes chromosome instability such as end-to-end fusion or rearrangement of chromosomes, thus resulting in subsequent carcinogenesis [5]. The associations of telomere length with cancer development and progression have been well investigated [6]. A series of experimental studies have identified shorter telomere length in tumor tissues, comparing with the corresponding adjacent nontumor tissues [6]. Meanwhile, recent studies have demonstrated that abnormal alternation (shortening or lengthening) of telomere length in peripheral blood leukocytes (PBLs) is significantly associated with the risk of various malignancies, including CRC [7]. In addition, several studies have reported that patients with longer leukocyte telomere length have worse overall survival (OS) of breast, kidney and liver cancer [8–10]. However, to date, whether leukocyte telomere length can predict CRC patients’ outcomes has never been studied. In this study, we measured the relative telomere length (RTL) of PBLs and assessed its prognostic value in CRC patients undergoing surgical resection. Furthermore, we explored the potential mechanisms underlying the association between telomere length and the survival of CRC patients. To the best of our knowledge, this is the first study to investigate the role of leukocyte telomere length in predicting CRC prognosis.
study was approved by the Ethic Committee of Fourth Military Medical University and informed consents were obtained from all participants.
original articles
Annals of Oncology
The mean inter-assay coefficient of variation (CV) was 6.4% (range, 3.2–9.1%), and the mean intra-assay CV was 3.9% (range, 2.1–6.8%), indicating good assay reproducibility. The median RTL in the training, testing and validation sets was 0.66, 0.68 and 0.64, respectively (Supplementary Figure S1, available at Annals of Oncology online). RTL was negatively correlated with age in all the three different subpopulations and the total population (Supplementary Figure S2, available at Annals of Oncology online). There was no significant association between RTL and most clinical characteristics of patients (Table 1). However, patients who died or developed relapse had shorter RTL than those who was alive or had no relapse in the three patient sets (P < 0.05 for all).
prognostic analysis of RTL in CRC patients
Table 1. Clinical characteristics of patients according to the RTL in the training, testing and validation sets Characteristics
Training set (n = 90) Short RTL Long RTL (n = 45) (n = 45)
Age (years) 61.87 (11.75) Sex Male 29 (64.4%) Female 16 (35.6%) Tumor location Colon 19 (42.2%) Rectum 26 (57.8%) TNM stage 0/I 13 (28.9%) II 13 (28.9%) III 14 (31.1%) IV 5 (11.1%) Differentiation Well 25 (55.6%) Moderate + Poor 20 (44.4%) Adjuvant chemotherapy No 21 (46.7%) Yes 24 (53.3%) Relapse Yes 32 (71.1%) No 13 (28.9%) Death Yes 27 (60.0%) No 18 (40.0%)
P-value
Testing set (n = 86) Short RTL Long RTL (n = 42) (n = 44)
P-value
Validation set (n = 395) Short RTL Long RTL (n = 251) (n = 144)
P-value
59.964 (10.59)
0.420b
60.33 (14.38)
54.79 (12.32)
0.058b
58.98 (12.21)
56.45 (12.00)
0.065b
22 (48.9%) 23 (51.1%)
0.136a
22 (52.4%) 20 (47.6%)
27 (61.4%) 17 (38.6%)
0.400a
137 (53.8%) 114 (46.2%)
79 (53.8%) 65 (46.2%)
0.123a
15 (33.3%) 30 (66.7%)
0.384a
19 (45.2%) 23 (54.8%)
17 (38.6%) 27 (61.4%)
0.535a
125 (49.8%) 126 (50.2%)
71 (49.1%) 73 (50.9%)
0.896a
9 (20.0%) 23 (51.1%) 11 (24.4%) 2 (4.5%)
0.161a
10 (23.8%) 16 (38.1%) 12 (28.6%) 4 (9.5%)
10 (22.7%) 19 (43.2%) 12 (27.3%) 3 (6.8%)
0.950a
38 (15.1%) 114 (45.4%) 83 (33.1%) 16 (6.4%)
22 (15.3%) 75 (52.1%) 41 (28.5%) 6 (4.1%)
0.519a
19 (42.2%) 26 (57.8%)
0.206a
23 (54.8%) 19 (45.2%)
18 (40.9%) 26 (59.1%)
0.199a
66 (26.3%) 185 (73.7%)
43 (29.9%) 101 (70.1%)
0.168a
18 (40.0%) 27 (60.0%)
0.523a
16 (38.1%) 26 (61.9%)
15 (34.1%) 29 (65.9%)
0.699a
84 (33.5%) 167 (66.5%)
56 (38.9%) 88 (61.1%)
0.278a
12 (26.7%) 33 (73.3%)
<0.001c
23 (54.8%) 19 (45.2%)
14 (31.8%) 30 (78.2%)
0.020c
69 (25.6%) 182 (74.4%)
36 (25.0%) 108 (75.0%)
0.005c
11 (24.4%) 34 (75.6%)
0.001c
20 (47.6%) 22 (52.4%)
11 (25.0%) 33 (75.0%)
0.007c
51 (20.3%) 200 (79.7%)
30 (20.8%) 114 (79.2%)
<0.001c
RTL, relative telomere length. Data are the mean (SD) or n (%), unless otherwise stated. a The P-values were calculated using a Pearson chi-square test. b The P-values were calculated using an unpaired Student’s t-test. c The P-values were calculated using the log-rank test.
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To predict the OS and relapse-free survival (RFS) with the best sensitivity and specificity, the optimal cutoff point of RTL in the training set (0.704 for both OS and RFS) was determined by ROC curves and then used to dichotomize CRC patients into either long or short RTL subgroups in all subsequent analyses (Supplementary Figure S3, available at Annals of Oncology online). We first explored the prognostic value of RTL in the
training set and found that patients with short RTL had poorer OS and RFS than those with long RTL (Figure 1A and B, P = 0.001 and P < 0.001, respectively). We then examined the effects of RTL on the survival of CRC patients in the internal testing set (Figure 1C and D). As expected, patients with short RTL in the testing set also showed worse OS and RFS than those with long RTL (P = 0.007 and P = 0.020, respectively). We further validated these findings in an independent CRC patient cohort (validation set) and found that patients with short RTL was confirmed to have shorter OS and RFS (Figure 1E and F, P < 0.001 and P = 0.005, respectively). In the combined patient population (Figure 1G and H), consistent results were obtained (P < 0.001 for both OS and RFS). Multivariate analysis confirmed that TNM stage, adjuvant chemotherapy and RTL were independent prognostic factors for both OS and RFS of CRC patients in the training, testing and validation sets (Table 2). When combined three sets, the significant results were consistently observed, showing that patients with short RTL had a 2.43-fold (95% CI, 1.53–3.45; P = 0.004) death risk increment and 2.26-fold (95% CI, 1.35–3.23; P = 0.009) relapse risk increment than those with long RTL (Table 2). Furthermore, we divided the patients into 10 subgroups by the tenciles of RTL and found that the risks of both
original articles A
Annals of Oncology
B
Training set 1.0
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n = 45
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0
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40
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0
10
20
30
40
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Figure 1. Kaplan–Meier curves of OS and RFS of CRC patients in the training (A and B), testing (C and D), validation (E and F) and total (G and H) populations.
| Chen et al.
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n = 44
0.2
Overall survival
50
1.0
0.8
Overall survival
Long RTL Short RTL
Log rank P < 0.001
0.0
Overall survival
Training set 1.0
original articles
Annals of Oncology
Table 2. Multivariable Cox regression analysis of CRC patients’ prognosis in training, testing and validation sets Variables
Testing set HR (95% CI)
P-value
Validation set HR (95% CI)
1.03 (0.54–1.96) 0.930 1.58 (0.83–3.02) 0.166 1.32 (0.67–2.59) 0.417 2.57 (1.68–4.63) <0.001 1.08 (0.53–2.22) 0.828
1.09 (0.52–2.30) 1.98 (0.83–4.77) 1.19 (0.55–2.55) 2.36 (1.27–7.44) 1.35 (0.56–3.21)
0.822 0.124 0.660 0.009 0.504
1.12 (0.73–1.73) 0.608 1.35 (0.89–2.04) 0.151 1.22 (0.81–1.85) 0.336 2.42 (1.61–3.64) <0.001 1.06 (0.61–1.82) 0.844
1.24 (0.90–1.72) 0.191 0.77 (0.55–1.07) 0.113 1.11 (0.80–1.53) 0.528 2.92 (1.94–4.41) <0.001 1.09 (0.75–1.58) 0.642
2.18 (1.16–4.46) 0.22 (0.07–0.69)
0.012 0.009
3.03 (1.35–6.78) 0.007 0.41 (0.18–0.97) 0.043
1.76 (1.17–2.65) 0.39 (0.22–0.70)
0.006 0.001
2.43 (1.53–3.45) 0.004 0.46 (0.32–0.66) <0.001
0.97 (0.53–1.79) 1.21 (0.67–2.17) 1.30 (0.70–2.41) 2.38 (1.35–6.71) 1.05 (0.55–2.01)
0.933 0.541 0.409 0.001 0.874
1.29 (0.65–2.55) 1.49 (0.64–2.58) 1.39 (0.69–2.79) 2.57 (1.38–6.91) 1.41 (0.64–3.10)
0.464 0.262 0.353 0.002 0.398
1.15 (0.79–1.68) 0.464 1.34 (0.93–1.93) 0.107 1.35 (0.93–1.94) 0.106 2.33 (1.63–3.34) <0.001 1.03 (0.65–1.65) 0.889
1.30 (0.97–1.73) 0.083 0.77 (0.58–1.03) 0.080 1.12 (0.84–1.49) 0.459 2.67 (1.87–3.82) <0.001 1.08 (0.78–1.49) 0.661
2.15 (1.14–4.17) 0.29 (0.11–0.76)
0.018 0.012
3.02 (1.47–6.22) 0.003 0.17 (0.06–0.52) 0.002
1.95 (1.36–2.80) <0.001 0.39 (0.23–0.65) <0.001
2.26 (1.35–3.23) 0.60 (0.44–0.82)
P-value
P-value
Total HR (95% CI)
P-value
0.009 0.002
95% CI, 95% confidence interval; CRC, colorectal cancer; HR, hazard ratio; OS, overall survival; RFS, relapse-free survival; RTL, relative telomere length. Hazard ratios and P-values were calculated by the multivariate Cox proportional hazards regression model, adjusted for age, sex, tumor location, TNM stage, differentiation, RTL and chemotherapy as covariates. a Only including stage II and III CRC patients.
death and relapse were increased when the RTL decreased with a dose-dependent manner (Supplementary Figure S4, available at Annals of Oncology online). In addition, when stratified by RTL, all patient subgroups had similar OS and RFS benefit from adjuvant chemotherapy (Supplementary Figure S5, available at Annals of Oncology online), indicating no predictive value of RTL for the response to adjuvant chemotherapy.
prognostic prediction of RTL complementing to TNM stage Considering prognosis heterogeneity in the same TNM stage, we assessed the prognostic value of RTL in different TNM stages in the combined patient populations. As shown in Supplementary Figure S6, available at Annals of Oncology online, short RTL was associated with both worse OS and RFS in all TNM stages. Furthermore, we evaluated whether the combination of RTL and TNM stage would improve survival prediction. Our ROC analysis showed that the area under curve for OS and RFS in the combination model of TNM stage and RTL was larger than that in either TNM stage or RTL alone model, indicating a better predicting efficacy (Figure 2A and B). In addition, Kaplan–Meier analyses showed that patients with long RTL at TNM stage I/II had the best OS and RFS, whereas those with short RTL at TNM stage III/IV had the worst OS and RFS (P < 0.001 for both OS and RFS; Figure 2C and D). All these data suggest that the combination
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use of TNM stage and RTL had a better efficacy in predicting CRC prognosis
immunophenotypes of lymphocytes and concentration of plasma cytokines in CRC patients with different RTL To explore the underlying mechanisms that account for the different prognosis of CRC patients with the different RTL, we examined the subtypes of lymphocytes in PBMCs and plasma cytokine concentration from additional 24 CRC patients. Our results showed that patients with short RTL had significantly higher frequency of CD4+ T cells (42.5 versus 38%, P = 0.019, Figure 3A) and lower frequency of B cells (6 versus 11%, P = 0.014, Figure 3C) than those with long RTL. In addition, patients with short RTL had significantly lower concentration of TGF-β1 than those with long RTL (6.2 versus 10.5 pg/ml, P = 0.043, Figure 3E). No significant difference on the percentage of other immune cells or cytokine concentration examined was observed between the two patient subgroups (Figure 3 and Supplementary Figure S7, available at Annals of Oncology online).
discussion In this study, we assessed the prognostic value of leukocyte telomere length in 571 CRC patients. We found that patients with short RTL showed poorer OS and RFS and RTL could serve as
doi:10.1093/annonc/mdu016 |
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OS Age (≥ 60 versus <60) Sex (male versus female) Location (rectum versus colon) TNM stage (III + IV versus 0/I + II) Differentiation (Moderate + Poor versus Well) RTL (short versus long) Adjuvant chemotherapya (yes versus no) RFS Age (≥ 60 versus <60) Sex (male versus female) Location (rectum versus colon) TNM stage (III + IV versus 0/I + II) Differentiation (Moderate + Poor versus Well) RTL (short versus long) Adjuvant chemotherapya (yes versus no)
Training set HR (95% CI)
original articles
Annals of Oncology
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B 1.0
1.0 RFS RTL+TNM RTL TNM Reference
OS RTL+TNM RTL TNM Reference
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n = 146 Long RTL+StageI/II reference Long RTL+StageIII/IV HR 2.97, 95% CI 1.67–5.28 Short RTL+StageI/II HR 2.24, 95% CI 1.33–3.79 Short RTL+StageIII/IV HR 6.05, 95% CI 3.64–10.05
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RTL+TNM 0.708 0.665–0.751 0.001 0.633 0.587–0.679 0.001 RTL 0.644 0.597–0.692 0.001 TNM
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Figure 2. Joint prognostic value of RTL and TNM stage. ROC curve analysis showed that RTL+TNM had a better prediction value than did TNM or RTL alone in both OS (A) and RFS (B). (C and D) Kaplan–Meier curves of OS and RFS of CRC patients subgrouped by RTL and TNM. Hazard ratios and 95% CIs were calculated by the multivariate Cox proportional hazards regression model, adjusted for age, sex, tumor location, differentiation, RTL and chemotherapy as covariates.
an independent prognostic factor for CRC patients. Furthermore, the combination of RTL and TNM stage significantly improved the prognosis prediction efficacy. In addition, short telomere length was significantly associated with higher CD4+ T cell percentage, lower B cell percentage in PBMCs and lower plasma TGF-β1 concentration. These findings suggest that RTL plays an important role in the progression of CRC possibly by the abnormal alternation of immune functions. As an indicator of biological age, telomere length has long been noted in its association with lifespan and aging-related diseases, such as cancers [12]. Our results further confirmed previous findings that there is a negative correlation between leukocyte telomere length and age [4]. A number of epidemiological studies have demonstrated that leukocyte telomere length is associated with risks of various cancers, including CRC [13].
| Chen et al.
A previous study based on in situ telomere length assessment has showed that telomere attrition occurs early during the carcinogenesis of colorectal epithelia [14]. In consistence with these findings, our study confirmed the significant association between leukocyte telomere length and clinical outcomes in CRC patients, which further indicates the important biological role of telomere dysfunction in cancer development and progression. The biological mechanism underlying the effects of RTL on CRC prognosis remains to be elucidated. One explanation is that short telomere accelerates senescence of cells, including immune cells [15]. Leukocyte telomere length may serve as an indicator of immune response capacity and cell replication history [16]. It has been reported that accelerated telomere erosion is associated with the declining of immune functions
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0.0
0.4
0.2
RTL+TNM 0.719 0.676–0.763 0.001 0.640 0.593–0.686 0.001 RTL 0.653 0.604–0.702 0.001 TNM
0.0
0.6
original articles
Annals of Oncology
B
C
P = 0.019 80 CD8+ T cells/PBMC (%)
40
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P = 0.043
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F P = 0.148
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Long RTL (n = 12)
Short RTL (n = 12)
Long RTL (n = 12)
Figure 3. Immune phenotype and plasma cytokine concentrations in CRC patients. (A–C) Flow cytometry detection of the frequency of CD4+, CD8+ and B cells in PBMCs of CRC patients with low and high RTLs (both n = 12). (D–F) Plasma concentrations of IL-2, TGF-β1 and IFN-γ were measured by ELISA in CRC patients with low and high RTLs (both n = 12).
[17]. In this study, we found that short telomere length was significantly associated with higher proportion of CD4+ T cells in PBMCs. These data gave rise to an assumption that CD4+ T cells might experience more clonal expansion in CRC patients with short RTL than those with long RTL and thus exhibited an increased degree of immune senescence as previous data suggested [15]. In addition to T cells, B cells are reported to play an important role in anticancer immune response by antigen presenting and cytotoxicity [18]. In our study, we found a low percentage of B cells in patients with short RTL, which is in consistent with previous report that immunosenescent individuals had decreased B cell number in the PBMCs [15]. Our data suggest the potential involvement of insufficient B cell immune function in poor CRC prognosis conferred by short RTL. Proper concentration of TGF-β1 plays an important role in preventing T cell apoptosis and promoting T cell expansion and the survival of memory T cells [19]. Additionally, TGF-β1 promotes the affinity maturation and secretion of antibodies in B cells [20]. Recent studies also demonstrate that individuals who suffer from autoimmune diseases have low TGF-β1 concentration and increased risk of malignancies, indicating the important role of TGF-β1 in the maintenance of normal immune functions [21]. In the present study, we found that patients with short RTL had a
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low concentration of TGF-β1, indicating a more extensive effect of telomere length on immune system. Collectively, all abovementioned findings support an assumption that short RTL might be a causative factor leading to insufficient immune capacity by affecting lymphocyte phenotypes and cytokine secretion, thus contributing to poor prognosis in CRC patients. However, we recognized that immune-related analyses were only performed in a small population of CRC patients (n = 24), which would limit the accuracy and generalization of our results and thus need further validation in future studies. Three previous studies have reported the opposite results, indicating that long telomere length in PBLs is associated with a worse prognosis in breast, kidney and liver cancer patients [8– 10]. Interestingly, two of these studies have also shown that liver and kidney cancer patients with long leukocyte RTL had higher levels of regulatory T cells (Tregs) [9, 10, 22], an important T cell subset that suppresses anticancer immune reaction [23]. In contrast, our study indicated no significant difference in Treg frequency among CRC patients with different RTLs. These contradictory findings suggest that telomere length may affect the immune functions of patients in a disease-specific manner. Further investigations are needed to elucidate the mechanisms underlying effects of RTL on cancer patient prognosis and immune functions.
doi:10.1093/annonc/mdu016 |
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P = 0.665
20
Long RTL (n = 12)
Short RTL (n = 12)
Long RTL (n = 12)
0.6 Plasma IL-2 level (pg/ml)
40
0
0
D
60
P = 0.014 30
Plasma IFN-g level (pg/ml)
CD4+ T cells/PBMC (%)
60
P = 0.686
B cells/PBMC (%)
A
original articles
funding This work was supported by Program for New Century Excellent Talents in University; National Natural Science Foundation (81171966 to JX) and National Key Technologies R&D Program (2011ZX09307-001-04 to JX) of China.
disclosure The authors have declared no conflicts of interest.
references 1. Jemal A, Bray F, Center MM et al. Global cancer statistics. CA Cancer J Clin 2011; 61: 69–90. 2. Shia J, Klimstra DS, Bagci P et al. TNM staging of colorectal carcinoma: issues and caveats. Semin Diagn Pathol 2012; 29: 142–153. 3. Xu L, Li S, Stohr BA. The role of telomere biology in cancer. Annu Rev Pathol 2013; 8: 49–78. 4. Bekaert S, De Meyer T, Van Oostveldt P. Telomere attrition as ageing biomarker. Anticancer Res 2005; 25: 3011–3021.
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5. Rampazzo E, Bertorelle R, Serra L et al. Relationship between telomere shortening, genetic instability, and site of tumour origin in colorectal cancers. Br J Cancer 2010; 102: 1300–1305. 6. Svenson U, Roos G. Telomere length as a biological marker in malignancy. Biochim Biophys Acta 2009; 1792: 317–323. 7. Hou L, Zhang X, Gawron AJ et al. Surrogate tissue telomere length and cancer risk: shorter or longer? Cancer Lett 2012; 319: 130–135. 8. Svenson U, Nordfjall K, Stegmayr B et al. Breast cancer survival is associated with telomere length in peripheral blood cells. Cancer Res 2008; 68: 3618–3623. 9. Svenson U, Ljungberg B, Roos G. Telomere length in peripheral blood predicts survival in clear cell renal cell carcinoma. Cancer Res 2009; 69: 2896–2901. 10. Liu HQ, An JZ, Liu J et al. Leukocyte telomere length predicts overall survival in hepatocellular carcinoma treated with transarterial chemoembolization. Carcinogenesis 2012; 33: 1040–1045. 11. Zlobec I, Vuong T, Hayashi S et al. A simple and reproducible scoring system for EGFR in colorectal cancer: application to prognosis and prediction of response to preoperative brachytherapy. Br J Cancer 2007; 96: 793–800. 12. Kong CM, Lee XW, Wang X. Telomere shortening in human diseases. FEBS J 2013; 280: 3180–3193. 13. Wentzensen IM, Mirabello L, Pfeiffer RM et al. The association of telomere length and cancer: a meta-analysis. Cancer Epidemiol Biomarkers Prev 2011; 20: 1238–1250. 14. Meeker AK, Hicks JL, Iacobuzio-Donahue CA et al. Telomere length abnormalities occur early in the initiation of epithelial carcinogenesis. Clin Cancer Res 2004; 10: 3317–3326. 15. Kaszubowska L. Telomere shortening and ageing of the immune system. J Physiol Pharmacol 2008; 59 (Suppl 9): 169–186. 16. Iancu EM, Speiser DE, Rufer N. Assessing ageing of individual T lymphocytes: mission impossible? Mech Ageing Dev 2008; 129: 67–78. 17. Damjanovic AK, Yang Y, Glaser R et al. Accelerated telomere erosion is associated with a declining immune function of caregivers of Alzheimer’s disease patients. J Immunol 2007; 179: 4249–4254. 18. Namm JP, Li Q, Lao X et al. B lymphocytes as effector cells in the immunotherapy of cancer. J Surg Oncol 2012; 105: 431–435. 19. Sung JL, Lin JT, Gorham JD. CD28 co-stimulation regulates the effect of transforming growth factor-beta1 on the proliferation of naive CD4+ T cells. Int Immunopharmacol 2003; 3: 233–245. 20. Snapper CM, Waegell W, Beernink H et al. Transforming growth factor-beta 1 is required for secretion of IgG of all subclasses by LPS-activated murine B cells in vitro. J Immunol 1993; 151: 4625–4636. 21. Malaguarnera M, Cristaldi E, Romano G et al. Autoimmunity in the elderly: implications for cancer. J Cancer Res Ther 2012; 8: 520–527. 22. Svenson U, Gronlund E, Soderstrom I et al. Telomere length in relation to immunological parameters in patients with renal cell carcinoma. PLoS One 2013; 8: e55543. 23. Rabinovich GA, Gabrilovich D, Sotomayor EM. Immunosuppressive strategies that are mediated by tumor cells. Ann Rev Immunol 2007; 25: 267–296. 24. Roth AD, Delorenzi M, Tejpar S et al. Integrated analysis of molecular and clinical prognostic factors in stage II/III colon cancer. J Natl Cancer Inst 2012; 104: 1635–1646.
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In clinical practice, TNM stage is widely used to predict the prognosis and assist treatment decision-making in CRC [2]. However, due to the clinical and molecular heterogeneity of CRC, patients in the same stage undergoing same therapeutic regimen often show remarkably different clinical outcomes [24]. Therefore, the combined use of molecular biomarkers and TNM stage has been carried out to improve the prognosis prediction of CRC. In this study, we identified RTL as an independent prognostic factor for both OS and RFS in CRC patients. Therefore, we conducted a joint effect analysis to assess whether the combination of telomere length and TNM stage could improve prognosis prediction in CRC patients. Expectedly, our results showed that integration of RTL into TNM stage-based prognosis prediction models significantly improved the prediction efficacy for both OS and RFS in CRC patients. In summary, our study for the first time demonstrates that short RTL is significantly associated with poor clinical outcomes of CRC patients, which may involve an immune-related mechanism. Furthermore, the combination of RTL and TNM stage markedly improved the prognostic prediction efficacy. Our findings suggest that leukocyte telomere length may serve as a useful biomarker for assessing the immune functions and prognosis of CRC patients, which may improve decision-making of individualized CRC treatment.
Annals of Oncology
Annals of Oncology 21. Verwaal VJ, van Ruth S, Witkamp A et al. Long-term survival of peritoneal carcinomatosis of colorectal origin. Ann Surg Oncol 2005; 12: 65–71. 22. Younan R, Kusamura S, Baratti D et al. Morbidity, toxicity, and mortality classification systems in the local regional treatment of peritoneal surface malignancy. J Surg Oncol 2008; 98: 253–257. 23. Cao C, Yan TD, Black D et al. A systematic review and meta-analysis of cytoreductive surgery with perioperative intraperitoneal chemotherapy for peritoneal carcinomatosis of colorectal origin. Ann Surg Oncol 2009; 16: 2152–2165. 24. Oosterling SJ, van der Bij GJ, van EM et al. Surgical trauma and peritoneal recurrence of colorectal carcinoma. Eur J Surg Oncol 2005; 31: 29–37. 25. Raa ST, Oosterling SJ, van der Kaaij NP et al. Surgery promotes implantation of disseminated tumor cells, but does not increase growth of tumor cell clusters. J Surg Oncol 2005; 92: 124–129.
26. van den Tol MP, ten RS, van Grevenstein WM et al. The post-surgical inflammatory response provokes enhanced tumour recurrence: a crucial role for neutrophils. Dig Surg 2007; 24: 388–394. 27. Gur I, Diggs BS, Wagner JA et al. Safety and outcomes following resection of colorectal liver metastases in the era of current perioperative chemotherapy. J Gastrointest Surg 2013; 17: 2133–2142. 28. Nordlinger B, Sorbye H, Glimelius B et al. Perioperative FOLFOX4 chemotherapy and surgery versus surgery alone for resectable liver metastases from colorectal cancer (EORTC 40983): long-term results of a randomised, controlled, phase 3 trial. Lancet Oncol 2013; 14: 1208–1215. 29. Passot G, Vaudoyer D, Cotte E et al. Progression following neoadjuvant systemic chemotherapy may not be a contraindication to a curative approach for colorectal carcinomatosis. Ann Surg 2012; 256: 125–129.
Annals of Oncology 25: 869–876, 2014 doi:10.1093/annonc/mdu016 Published online 10 March 2014
Y. Chen1, †, F. Qu1, †, X. He2, G. Bao2, X. Liu3, S. Wan4 & J. Xing1* 1 State Key Laboratory of Cancer Biology and Experimental Teaching Center of Basic Medicine, Fourth Military Medical University, Xi’an; 2Department of General Surgery, Tangdu Hospital, Fourth Military Medical University, Xi’an; 3Deparment of Gastroenterology, Xijing Hospital of Digestive Disease, Fourth Military Medical University, Xi’an; 4 Pharmaceutical College, Henan University, Kaifeng, People’s Republic of China
Received 15 October 2013; revised 19 December 2013; accepted 30 December 2013
Background: Numerous studies indicate that the leukocyte telomere length is associated with the risk of cancers, including colorectal cancer (CRC). However, the prognostic value of leukocyte telomere length in CRC patients has not been investigated. Patients and methods: Relative telomere length (RTL) of peripheral blood leukocytes (PBLs) from 571 CRC patients receiving surgical resection was measured using a polymerase chain reaction-based method. The Cox proportional hazards ratio model and the Kaplan–Meier curve were used to estimate the association between RTL and the clinical outcome of CRC patients in the training set (90 patients) and the testing set (86 patients). Finally, an independent cohort of 395 patients was used as an external validation set. The immunophenotype of PBLs and the plasma concentration of several immune-related cytokines were determined by flow cytometry and enzyme-linked immunosorbent assay, respectively. Results: Patients with shorter RTL had significantly poorer overall survival and relapse-free survival than those with longer RTL in the training, testing and validation sets. Furthermore, leukocyte RTL and Tumor-Node-Metastasis (TNM) stage exhibited a significant joint effect in the prognosis prediction of combined CRC patients, indicating that patients with both short RTL and advanced stages had the worst prognosis, when compared with other subgroups. In addition, patients with short RTL showed the higher percentage of CD4+ T cell and the lower percentage of B cell in peripheral blood mononuclear cells, as well as the lower concentration of plasma transforming growth factor-β1, suggesting a possibility that the immune functions changed with RTL alteration. Conclusions: Our study for the first time demonstrates that leukocyte RTL is an independent prognostic marker complementing TNM stage and associated with the immune functions in CRC patients.
*Correspondence to: Dr Jinliang Xing, State Key Laboratory of Cancer Biology and Experimental Teaching Center of Basic Medicine, Fourth Military Medical University; 169 Changle West Road, Xi’an 710032, People’s Republic of China. Tel: +86-29-84774764; Fax: +86-29-84774764; E-mail: [email protected] †
YC and FQ contributed equally to this work.
© The Author 2014. Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved. For permissions, please email: [email protected].
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Short leukocyte telomere length predicts poor prognosis and indicates altered immune functions in colorectal cancer patients
original articles
Annals of Oncology
Key words: colorectal cancer, cytokine, immunophenotype, leukocyte relative telomere length, overall survival, relapse-free survival
introduction
patients and methods patients and samples A total of 571 eligible patients with primary colorectal adenocarcinoma were enrolled from two independent sites, Xijing Hospital and Tangdu Hospital, Xi’an, China. All patients received surgical resection and had no preoperative anticancer treatment. Patient subgrouping was described in Supplementary Methods, available at Annals of Oncology online. Twenty-four additional CRC patients were enrolled from Xijing Hospital for immunoassays. Before surgery, 5 ml of venous blood sample was collected from each patient. The
| Chen et al.
measurement of RTL by real-time polymerase chain reaction Leukocyte genomic DNA was extracted from blood samples using the RelaxGene Blood DNA System (TIANGEN, Beijing, China) according to the manufacturer’s instructions. RTL was measured using a real-time polymerase chain reaction-based method detailedly described in Supplementary Methods, available at Annals of Oncology online.
determination of lymphocyte immunophenotype by flow cytometery Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation over Ficoll-Hypaque (Amersham Pharmacia Biotech, NJ, USA) from venous blood of additional 24 CRC patients. The cells were then fixed and stained with isotype control immunoglobulins or fluorescenceconjugated antibodies against the following immune markers: CD3, CD4, CD8, CD25, FOXP3, CD19 and CD56 (eBioscience, San Diego, CA, USA). Finally, the subtype analysis of PBMCs was carried out on a FACScan flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA).
cytokine detection by enzyme-linked immunosorbent assay Blood plasma from the same 24 CRC patients was isolated by centrifugation at 2800 ×g under 4°C. The concentrations of interleukin (IL)-2, IL-4, transforming growth factor (TGF)-β1, tumor necrosis factor (TNF)-α and interferon (IFN)-γ were examined by enzyme-linked immunosorbent assay (ELISA) using commercial kits (eBioscience) according to the manufacturer’s instructions.
statistical analysis The difference of variables among subgroups was compared by the corresponding statistical methods. More details were described in Supplementary Methods, available at Annals of Oncology online. Receiver operating characteristic (ROC) curve was used to calculate the optimal cutoff point of RTL for prognostic prediction in the training set as described previously [11]. The multivariate Cox proportional hazards regression model and the Kaplan–Meier survival curve were used for prognosis evaluation. All statistical analyses were performed using the SPSS Statistics 19.0 software (IBM), and P < 0.05 was considered statistically significant.
results characteristics and RTL distribution of CRC patients The clinical characteristics of the study populations were summarized in Supplementary Table S1, available at Annals of Oncology online. Overall, there were 361 patients who received standard fluorouracil (FU)-based adjuvant chemotherapy (98 with CapeOX regimen, 252 with FOLFOX regimen and 11 with capecitabine single-treatment). One hundred and fifty patients died of CRC and 186 developed relapse during the follow-up period (median, 28 months; range, 6–60 months).
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Colorectal cancer (CRC) is one of the most common malignancies worldwide, with over 600 000 deaths each year [1]. In recent years, increased incidence and death rate of CRC is observed in several regions where this disease has previously been reported to be at low risk. Tumor-Node-Metastasis (TNM) staging system is widely used to assist the decision-making of treatment and prognosis predicting in CRC patients [2]. However, due to the molecular and genetic heterogeneity, TNM stage alone is not sufficient to accurately predict the clinical outcomes of CRC patients. Therefore, it is urgent to explore new biomarkers to complement TNM staging system for better prediction of the CRC prognosis. Telomere is a specified structure consisting of tandem repeats of TTAGGG which caps the end of linear chromosomes in eukaryote cells [3]. Telomere plays a critical role in prevention of genomic instability and DNA shortening-induced damage responses. Under physiological conditions, most somatic cells suffer from gradually shortening of telomere by 15–50 bp each year due to the lack of telomerase activity [4]. Abnormal alteration of telomere length causes chromosome instability such as end-to-end fusion or rearrangement of chromosomes, thus resulting in subsequent carcinogenesis [5]. The associations of telomere length with cancer development and progression have been well investigated [6]. A series of experimental studies have identified shorter telomere length in tumor tissues, comparing with the corresponding adjacent nontumor tissues [6]. Meanwhile, recent studies have demonstrated that abnormal alternation (shortening or lengthening) of telomere length in peripheral blood leukocytes (PBLs) is significantly associated with the risk of various malignancies, including CRC [7]. In addition, several studies have reported that patients with longer leukocyte telomere length have worse overall survival (OS) of breast, kidney and liver cancer [8–10]. However, to date, whether leukocyte telomere length can predict CRC patients’ outcomes has never been studied. In this study, we measured the relative telomere length (RTL) of PBLs and assessed its prognostic value in CRC patients undergoing surgical resection. Furthermore, we explored the potential mechanisms underlying the association between telomere length and the survival of CRC patients. To the best of our knowledge, this is the first study to investigate the role of leukocyte telomere length in predicting CRC prognosis.
study was approved by the Ethic Committee of Fourth Military Medical University and informed consents were obtained from all participants.
original articles
Annals of Oncology
The mean inter-assay coefficient of variation (CV) was 6.4% (range, 3.2–9.1%), and the mean intra-assay CV was 3.9% (range, 2.1–6.8%), indicating good assay reproducibility. The median RTL in the training, testing and validation sets was 0.66, 0.68 and 0.64, respectively (Supplementary Figure S1, available at Annals of Oncology online). RTL was negatively correlated with age in all the three different subpopulations and the total population (Supplementary Figure S2, available at Annals of Oncology online). There was no significant association between RTL and most clinical characteristics of patients (Table 1). However, patients who died or developed relapse had shorter RTL than those who was alive or had no relapse in the three patient sets (P < 0.05 for all).
prognostic analysis of RTL in CRC patients
Table 1. Clinical characteristics of patients according to the RTL in the training, testing and validation sets Characteristics
Training set (n = 90) Short RTL Long RTL (n = 45) (n = 45)
Age (years) 61.87 (11.75) Sex Male 29 (64.4%) Female 16 (35.6%) Tumor location Colon 19 (42.2%) Rectum 26 (57.8%) TNM stage 0/I 13 (28.9%) II 13 (28.9%) III 14 (31.1%) IV 5 (11.1%) Differentiation Well 25 (55.6%) Moderate + Poor 20 (44.4%) Adjuvant chemotherapy No 21 (46.7%) Yes 24 (53.3%) Relapse Yes 32 (71.1%) No 13 (28.9%) Death Yes 27 (60.0%) No 18 (40.0%)
P-value
Testing set (n = 86) Short RTL Long RTL (n = 42) (n = 44)
P-value
Validation set (n = 395) Short RTL Long RTL (n = 251) (n = 144)
P-value
59.964 (10.59)
0.420b
60.33 (14.38)
54.79 (12.32)
0.058b
58.98 (12.21)
56.45 (12.00)
0.065b
22 (48.9%) 23 (51.1%)
0.136a
22 (52.4%) 20 (47.6%)
27 (61.4%) 17 (38.6%)
0.400a
137 (53.8%) 114 (46.2%)
79 (53.8%) 65 (46.2%)
0.123a
15 (33.3%) 30 (66.7%)
0.384a
19 (45.2%) 23 (54.8%)
17 (38.6%) 27 (61.4%)
0.535a
125 (49.8%) 126 (50.2%)
71 (49.1%) 73 (50.9%)
0.896a
9 (20.0%) 23 (51.1%) 11 (24.4%) 2 (4.5%)
0.161a
10 (23.8%) 16 (38.1%) 12 (28.6%) 4 (9.5%)
10 (22.7%) 19 (43.2%) 12 (27.3%) 3 (6.8%)
0.950a
38 (15.1%) 114 (45.4%) 83 (33.1%) 16 (6.4%)
22 (15.3%) 75 (52.1%) 41 (28.5%) 6 (4.1%)
0.519a
19 (42.2%) 26 (57.8%)
0.206a
23 (54.8%) 19 (45.2%)
18 (40.9%) 26 (59.1%)
0.199a
66 (26.3%) 185 (73.7%)
43 (29.9%) 101 (70.1%)
0.168a
18 (40.0%) 27 (60.0%)
0.523a
16 (38.1%) 26 (61.9%)
15 (34.1%) 29 (65.9%)
0.699a
84 (33.5%) 167 (66.5%)
56 (38.9%) 88 (61.1%)
0.278a
12 (26.7%) 33 (73.3%)
<0.001c
23 (54.8%) 19 (45.2%)
14 (31.8%) 30 (78.2%)
0.020c
69 (25.6%) 182 (74.4%)
36 (25.0%) 108 (75.0%)
0.005c
11 (24.4%) 34 (75.6%)
0.001c
20 (47.6%) 22 (52.4%)
11 (25.0%) 33 (75.0%)
0.007c
51 (20.3%) 200 (79.7%)
30 (20.8%) 114 (79.2%)
<0.001c
RTL, relative telomere length. Data are the mean (SD) or n (%), unless otherwise stated. a The P-values were calculated using a Pearson chi-square test. b The P-values were calculated using an unpaired Student’s t-test. c The P-values were calculated using the log-rank test.
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To predict the OS and relapse-free survival (RFS) with the best sensitivity and specificity, the optimal cutoff point of RTL in the training set (0.704 for both OS and RFS) was determined by ROC curves and then used to dichotomize CRC patients into either long or short RTL subgroups in all subsequent analyses (Supplementary Figure S3, available at Annals of Oncology online). We first explored the prognostic value of RTL in the
training set and found that patients with short RTL had poorer OS and RFS than those with long RTL (Figure 1A and B, P = 0.001 and P < 0.001, respectively). We then examined the effects of RTL on the survival of CRC patients in the internal testing set (Figure 1C and D). As expected, patients with short RTL in the testing set also showed worse OS and RFS than those with long RTL (P = 0.007 and P = 0.020, respectively). We further validated these findings in an independent CRC patient cohort (validation set) and found that patients with short RTL was confirmed to have shorter OS and RFS (Figure 1E and F, P < 0.001 and P = 0.005, respectively). In the combined patient population (Figure 1G and H), consistent results were obtained (P < 0.001 for both OS and RFS). Multivariate analysis confirmed that TNM stage, adjuvant chemotherapy and RTL were independent prognostic factors for both OS and RFS of CRC patients in the training, testing and validation sets (Table 2). When combined three sets, the significant results were consistently observed, showing that patients with short RTL had a 2.43-fold (95% CI, 1.53–3.45; P = 0.004) death risk increment and 2.26-fold (95% CI, 1.35–3.23; P = 0.009) relapse risk increment than those with long RTL (Table 2). Furthermore, we divided the patients into 10 subgroups by the tenciles of RTL and found that the risks of both
original articles A
Annals of Oncology
B
Training set 1.0
Overall survival
Relapse-free survival
n = 45
0.8 0.6
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Log rank P = 0.020
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Log rank P = 0.005
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Log rank P < 0.001 0.0
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0
10
20
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40
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0
10
20
30
40
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Figure 1. Kaplan–Meier curves of OS and RFS of CRC patients in the training (A and B), testing (C and D), validation (E and F) and total (G and H) populations.
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n = 44
0.2
Overall survival
50
1.0
0.8
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Long RTL Short RTL
Log rank P < 0.001
0.0
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Training set 1.0
original articles
Annals of Oncology
Table 2. Multivariable Cox regression analysis of CRC patients’ prognosis in training, testing and validation sets Variables
Testing set HR (95% CI)
P-value
Validation set HR (95% CI)
1.03 (0.54–1.96) 0.930 1.58 (0.83–3.02) 0.166 1.32 (0.67–2.59) 0.417 2.57 (1.68–4.63) <0.001 1.08 (0.53–2.22) 0.828
1.09 (0.52–2.30) 1.98 (0.83–4.77) 1.19 (0.55–2.55) 2.36 (1.27–7.44) 1.35 (0.56–3.21)
0.822 0.124 0.660 0.009 0.504
1.12 (0.73–1.73) 0.608 1.35 (0.89–2.04) 0.151 1.22 (0.81–1.85) 0.336 2.42 (1.61–3.64) <0.001 1.06 (0.61–1.82) 0.844
1.24 (0.90–1.72) 0.191 0.77 (0.55–1.07) 0.113 1.11 (0.80–1.53) 0.528 2.92 (1.94–4.41) <0.001 1.09 (0.75–1.58) 0.642
2.18 (1.16–4.46) 0.22 (0.07–0.69)
0.012 0.009
3.03 (1.35–6.78) 0.007 0.41 (0.18–0.97) 0.043
1.76 (1.17–2.65) 0.39 (0.22–0.70)
0.006 0.001
2.43 (1.53–3.45) 0.004 0.46 (0.32–0.66) <0.001
0.97 (0.53–1.79) 1.21 (0.67–2.17) 1.30 (0.70–2.41) 2.38 (1.35–6.71) 1.05 (0.55–2.01)
0.933 0.541 0.409 0.001 0.874
1.29 (0.65–2.55) 1.49 (0.64–2.58) 1.39 (0.69–2.79) 2.57 (1.38–6.91) 1.41 (0.64–3.10)
0.464 0.262 0.353 0.002 0.398
1.15 (0.79–1.68) 0.464 1.34 (0.93–1.93) 0.107 1.35 (0.93–1.94) 0.106 2.33 (1.63–3.34) <0.001 1.03 (0.65–1.65) 0.889
1.30 (0.97–1.73) 0.083 0.77 (0.58–1.03) 0.080 1.12 (0.84–1.49) 0.459 2.67 (1.87–3.82) <0.001 1.08 (0.78–1.49) 0.661
2.15 (1.14–4.17) 0.29 (0.11–0.76)
0.018 0.012
3.02 (1.47–6.22) 0.003 0.17 (0.06–0.52) 0.002
1.95 (1.36–2.80) <0.001 0.39 (0.23–0.65) <0.001
2.26 (1.35–3.23) 0.60 (0.44–0.82)
P-value
P-value
Total HR (95% CI)
P-value
0.009 0.002
95% CI, 95% confidence interval; CRC, colorectal cancer; HR, hazard ratio; OS, overall survival; RFS, relapse-free survival; RTL, relative telomere length. Hazard ratios and P-values were calculated by the multivariate Cox proportional hazards regression model, adjusted for age, sex, tumor location, TNM stage, differentiation, RTL and chemotherapy as covariates. a Only including stage II and III CRC patients.
death and relapse were increased when the RTL decreased with a dose-dependent manner (Supplementary Figure S4, available at Annals of Oncology online). In addition, when stratified by RTL, all patient subgroups had similar OS and RFS benefit from adjuvant chemotherapy (Supplementary Figure S5, available at Annals of Oncology online), indicating no predictive value of RTL for the response to adjuvant chemotherapy.
prognostic prediction of RTL complementing to TNM stage Considering prognosis heterogeneity in the same TNM stage, we assessed the prognostic value of RTL in different TNM stages in the combined patient populations. As shown in Supplementary Figure S6, available at Annals of Oncology online, short RTL was associated with both worse OS and RFS in all TNM stages. Furthermore, we evaluated whether the combination of RTL and TNM stage would improve survival prediction. Our ROC analysis showed that the area under curve for OS and RFS in the combination model of TNM stage and RTL was larger than that in either TNM stage or RTL alone model, indicating a better predicting efficacy (Figure 2A and B). In addition, Kaplan–Meier analyses showed that patients with long RTL at TNM stage I/II had the best OS and RFS, whereas those with short RTL at TNM stage III/IV had the worst OS and RFS (P < 0.001 for both OS and RFS; Figure 2C and D). All these data suggest that the combination
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use of TNM stage and RTL had a better efficacy in predicting CRC prognosis
immunophenotypes of lymphocytes and concentration of plasma cytokines in CRC patients with different RTL To explore the underlying mechanisms that account for the different prognosis of CRC patients with the different RTL, we examined the subtypes of lymphocytes in PBMCs and plasma cytokine concentration from additional 24 CRC patients. Our results showed that patients with short RTL had significantly higher frequency of CD4+ T cells (42.5 versus 38%, P = 0.019, Figure 3A) and lower frequency of B cells (6 versus 11%, P = 0.014, Figure 3C) than those with long RTL. In addition, patients with short RTL had significantly lower concentration of TGF-β1 than those with long RTL (6.2 versus 10.5 pg/ml, P = 0.043, Figure 3E). No significant difference on the percentage of other immune cells or cytokine concentration examined was observed between the two patient subgroups (Figure 3 and Supplementary Figure S7, available at Annals of Oncology online).
discussion In this study, we assessed the prognostic value of leukocyte telomere length in 571 CRC patients. We found that patients with short RTL showed poorer OS and RFS and RTL could serve as
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OS Age (≥ 60 versus <60) Sex (male versus female) Location (rectum versus colon) TNM stage (III + IV versus 0/I + II) Differentiation (Moderate + Poor versus Well) RTL (short versus long) Adjuvant chemotherapya (yes versus no) RFS Age (≥ 60 versus <60) Sex (male versus female) Location (rectum versus colon) TNM stage (III + IV versus 0/I + II) Differentiation (Moderate + Poor versus Well) RTL (short versus long) Adjuvant chemotherapya (yes versus no)
Training set HR (95% CI)
original articles
Annals of Oncology
A
B 1.0
1.0 RFS RTL+TNM RTL TNM Reference
OS RTL+TNM RTL TNM Reference
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0.6
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1 - Specificity
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D 1.0
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Log rank P < 0.001
Log rank P < 0.001 n = 136
0.8
n = 136
0.6
n = 226 n = 63
0.4
n = 146 Long RTL+StageI/II reference Long RTL+StageIII/IV HR 2.97, 95% CI 1.67–5.28 Short RTL+StageI/II HR 2.24, 95% CI 1.33–3.79 Short RTL+StageIII/IV HR 6.05, 95% CI 3.64–10.05
0.2 0.0 0
10
20
30
40
50
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Overall survival
95%Cl
RTL+TNM 0.708 0.665–0.751 0.001 0.633 0.587–0.679 0.001 RTL 0.644 0.597–0.692 0.001 TNM
0.8 n = 226 0.6 n = 63 n = 146
0.4 Long RTL+StageI/II Long RTL+StageIII/IV Short RTL+StageI/II Short RTL+StageIII/IV
0.2 0.0
60
Months
0
10
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reference HR 2.66, 95% CI 1.60–4.44 HR 1.78, 95% CI 1.12–2.79 HR 4.21, 95% CI 2.71–6.55
30
40
50
60
Months
Figure 2. Joint prognostic value of RTL and TNM stage. ROC curve analysis showed that RTL+TNM had a better prediction value than did TNM or RTL alone in both OS (A) and RFS (B). (C and D) Kaplan–Meier curves of OS and RFS of CRC patients subgrouped by RTL and TNM. Hazard ratios and 95% CIs were calculated by the multivariate Cox proportional hazards regression model, adjusted for age, sex, tumor location, differentiation, RTL and chemotherapy as covariates.
an independent prognostic factor for CRC patients. Furthermore, the combination of RTL and TNM stage significantly improved the prognosis prediction efficacy. In addition, short telomere length was significantly associated with higher CD4+ T cell percentage, lower B cell percentage in PBMCs and lower plasma TGF-β1 concentration. These findings suggest that RTL plays an important role in the progression of CRC possibly by the abnormal alternation of immune functions. As an indicator of biological age, telomere length has long been noted in its association with lifespan and aging-related diseases, such as cancers [12]. Our results further confirmed previous findings that there is a negative correlation between leukocyte telomere length and age [4]. A number of epidemiological studies have demonstrated that leukocyte telomere length is associated with risks of various cancers, including CRC [13].
| Chen et al.
A previous study based on in situ telomere length assessment has showed that telomere attrition occurs early during the carcinogenesis of colorectal epithelia [14]. In consistence with these findings, our study confirmed the significant association between leukocyte telomere length and clinical outcomes in CRC patients, which further indicates the important biological role of telomere dysfunction in cancer development and progression. The biological mechanism underlying the effects of RTL on CRC prognosis remains to be elucidated. One explanation is that short telomere accelerates senescence of cells, including immune cells [15]. Leukocyte telomere length may serve as an indicator of immune response capacity and cell replication history [16]. It has been reported that accelerated telomere erosion is associated with the declining of immune functions
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0.0
0.4
0.2
RTL+TNM 0.719 0.676–0.763 0.001 0.640 0.593–0.686 0.001 RTL 0.653 0.604–0.702 0.001 TNM
0.0
0.6
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Annals of Oncology
B
C
P = 0.019 80 CD8+ T cells/PBMC (%)
40
20
Short RTL (n = 12)
20
10
0
E
P = 0.043
Short RTL (n = 12)
F P = 0.148
20 Plasma TGF-b1 level (pg/ml)
0.4
0.2
1.0
15
10
5
0
0.0 Short RTL (n = 12)
Long RTL (n = 12)
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0.8 0.6 0.4 0.2 0.0
Short RTL (n = 12)
Long RTL (n = 12)
Short RTL (n = 12)
Long RTL (n = 12)
Figure 3. Immune phenotype and plasma cytokine concentrations in CRC patients. (A–C) Flow cytometry detection of the frequency of CD4+, CD8+ and B cells in PBMCs of CRC patients with low and high RTLs (both n = 12). (D–F) Plasma concentrations of IL-2, TGF-β1 and IFN-γ were measured by ELISA in CRC patients with low and high RTLs (both n = 12).
[17]. In this study, we found that short telomere length was significantly associated with higher proportion of CD4+ T cells in PBMCs. These data gave rise to an assumption that CD4+ T cells might experience more clonal expansion in CRC patients with short RTL than those with long RTL and thus exhibited an increased degree of immune senescence as previous data suggested [15]. In addition to T cells, B cells are reported to play an important role in anticancer immune response by antigen presenting and cytotoxicity [18]. In our study, we found a low percentage of B cells in patients with short RTL, which is in consistent with previous report that immunosenescent individuals had decreased B cell number in the PBMCs [15]. Our data suggest the potential involvement of insufficient B cell immune function in poor CRC prognosis conferred by short RTL. Proper concentration of TGF-β1 plays an important role in preventing T cell apoptosis and promoting T cell expansion and the survival of memory T cells [19]. Additionally, TGF-β1 promotes the affinity maturation and secretion of antibodies in B cells [20]. Recent studies also demonstrate that individuals who suffer from autoimmune diseases have low TGF-β1 concentration and increased risk of malignancies, indicating the important role of TGF-β1 in the maintenance of normal immune functions [21]. In the present study, we found that patients with short RTL had a
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low concentration of TGF-β1, indicating a more extensive effect of telomere length on immune system. Collectively, all abovementioned findings support an assumption that short RTL might be a causative factor leading to insufficient immune capacity by affecting lymphocyte phenotypes and cytokine secretion, thus contributing to poor prognosis in CRC patients. However, we recognized that immune-related analyses were only performed in a small population of CRC patients (n = 24), which would limit the accuracy and generalization of our results and thus need further validation in future studies. Three previous studies have reported the opposite results, indicating that long telomere length in PBLs is associated with a worse prognosis in breast, kidney and liver cancer patients [8– 10]. Interestingly, two of these studies have also shown that liver and kidney cancer patients with long leukocyte RTL had higher levels of regulatory T cells (Tregs) [9, 10, 22], an important T cell subset that suppresses anticancer immune reaction [23]. In contrast, our study indicated no significant difference in Treg frequency among CRC patients with different RTLs. These contradictory findings suggest that telomere length may affect the immune functions of patients in a disease-specific manner. Further investigations are needed to elucidate the mechanisms underlying effects of RTL on cancer patient prognosis and immune functions.
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P = 0.665
20
Long RTL (n = 12)
Short RTL (n = 12)
Long RTL (n = 12)
0.6 Plasma IL-2 level (pg/ml)
40
0
0
D
60
P = 0.014 30
Plasma IFN-g level (pg/ml)
CD4+ T cells/PBMC (%)
60
P = 0.686
B cells/PBMC (%)
A
original articles
funding This work was supported by Program for New Century Excellent Talents in University; National Natural Science Foundation (81171966 to JX) and National Key Technologies R&D Program (2011ZX09307-001-04 to JX) of China.
disclosure The authors have declared no conflicts of interest.
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5. Rampazzo E, Bertorelle R, Serra L et al. Relationship between telomere shortening, genetic instability, and site of tumour origin in colorectal cancers. Br J Cancer 2010; 102: 1300–1305. 6. Svenson U, Roos G. Telomere length as a biological marker in malignancy. Biochim Biophys Acta 2009; 1792: 317–323. 7. Hou L, Zhang X, Gawron AJ et al. Surrogate tissue telomere length and cancer risk: shorter or longer? Cancer Lett 2012; 319: 130–135. 8. Svenson U, Nordfjall K, Stegmayr B et al. Breast cancer survival is associated with telomere length in peripheral blood cells. Cancer Res 2008; 68: 3618–3623. 9. Svenson U, Ljungberg B, Roos G. Telomere length in peripheral blood predicts survival in clear cell renal cell carcinoma. Cancer Res 2009; 69: 2896–2901. 10. Liu HQ, An JZ, Liu J et al. Leukocyte telomere length predicts overall survival in hepatocellular carcinoma treated with transarterial chemoembolization. Carcinogenesis 2012; 33: 1040–1045. 11. Zlobec I, Vuong T, Hayashi S et al. A simple and reproducible scoring system for EGFR in colorectal cancer: application to prognosis and prediction of response to preoperative brachytherapy. Br J Cancer 2007; 96: 793–800. 12. Kong CM, Lee XW, Wang X. Telomere shortening in human diseases. FEBS J 2013; 280: 3180–3193. 13. Wentzensen IM, Mirabello L, Pfeiffer RM et al. The association of telomere length and cancer: a meta-analysis. Cancer Epidemiol Biomarkers Prev 2011; 20: 1238–1250. 14. Meeker AK, Hicks JL, Iacobuzio-Donahue CA et al. Telomere length abnormalities occur early in the initiation of epithelial carcinogenesis. Clin Cancer Res 2004; 10: 3317–3326. 15. Kaszubowska L. Telomere shortening and ageing of the immune system. J Physiol Pharmacol 2008; 59 (Suppl 9): 169–186. 16. Iancu EM, Speiser DE, Rufer N. Assessing ageing of individual T lymphocytes: mission impossible? Mech Ageing Dev 2008; 129: 67–78. 17. Damjanovic AK, Yang Y, Glaser R et al. Accelerated telomere erosion is associated with a declining immune function of caregivers of Alzheimer’s disease patients. J Immunol 2007; 179: 4249–4254. 18. Namm JP, Li Q, Lao X et al. B lymphocytes as effector cells in the immunotherapy of cancer. J Surg Oncol 2012; 105: 431–435. 19. Sung JL, Lin JT, Gorham JD. CD28 co-stimulation regulates the effect of transforming growth factor-beta1 on the proliferation of naive CD4+ T cells. Int Immunopharmacol 2003; 3: 233–245. 20. Snapper CM, Waegell W, Beernink H et al. Transforming growth factor-beta 1 is required for secretion of IgG of all subclasses by LPS-activated murine B cells in vitro. J Immunol 1993; 151: 4625–4636. 21. Malaguarnera M, Cristaldi E, Romano G et al. Autoimmunity in the elderly: implications for cancer. J Cancer Res Ther 2012; 8: 520–527. 22. Svenson U, Gronlund E, Soderstrom I et al. Telomere length in relation to immunological parameters in patients with renal cell carcinoma. PLoS One 2013; 8: e55543. 23. Rabinovich GA, Gabrilovich D, Sotomayor EM. Immunosuppressive strategies that are mediated by tumor cells. Ann Rev Immunol 2007; 25: 267–296. 24. Roth AD, Delorenzi M, Tejpar S et al. Integrated analysis of molecular and clinical prognostic factors in stage II/III colon cancer. J Natl Cancer Inst 2012; 104: 1635–1646.
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In clinical practice, TNM stage is widely used to predict the prognosis and assist treatment decision-making in CRC [2]. However, due to the clinical and molecular heterogeneity of CRC, patients in the same stage undergoing same therapeutic regimen often show remarkably different clinical outcomes [24]. Therefore, the combined use of molecular biomarkers and TNM stage has been carried out to improve the prognosis prediction of CRC. In this study, we identified RTL as an independent prognostic factor for both OS and RFS in CRC patients. Therefore, we conducted a joint effect analysis to assess whether the combination of telomere length and TNM stage could improve prognosis prediction in CRC patients. Expectedly, our results showed that integration of RTL into TNM stage-based prognosis prediction models significantly improved the prediction efficacy for both OS and RFS in CRC patients. In summary, our study for the first time demonstrates that short RTL is significantly associated with poor clinical outcomes of CRC patients, which may involve an immune-related mechanism. Furthermore, the combination of RTL and TNM stage markedly improved the prognostic prediction efficacy. Our findings suggest that leukocyte telomere length may serve as a useful biomarker for assessing the immune functions and prognosis of CRC patients, which may improve decision-making of individualized CRC treatment.
Annals of Oncology